Table 1.
Pathogenicity of Australian isolates of Leptosphaeria maculans on cotyledons of Brassica napus cultivars Q2 and Columbus and B. juncea cv. Aurea.
Table 2.
Alleles of AvrLm1, AvrLm6, LmCys1 and LmCys2 in 295 Australian isolates of Leptosphaeria maculans.
Table 3.
Changes in the frequency of the profile of alleles of AvrLm1 and Avrlm6 of Leptosphaeria maculans isolates before and after the breakdown of ‘sylvestris resistance’ and in relation to stubble source of isolates.
Figure 1.
Location of the genes and non-coding, non-repetitive regions analysed from a 520 kb region of the Leptosphaeria maculans genome located on a 2.6 Mb chromosome in isolate v23.1.3.
(A) Schematic representation of the AvrLm1-LmCys2 genomic region whereby highly-repetitive sequences with low GC content (red) flank single copy sections with high GC content (white). Four genes encoding small-secreted proteins, AvrLm1, AvrLm6, LmCys1 and LmCys2, were sequenced from 295 Australian isolates, whilst the remaining three genes and four non-coding, non-repetitive regions (NC1-4) were sequenced in 84 of the 295 isolates. Repeat-induced point (RIP) mutations were detected in AvrLm6, LmCys1, LmTrans and two non-coding, non-repetitive regions (NC3 and NC4) (black). (B) Location of single-copy sequences relative to the flanking repetitive regions. Within each of the two single copy regions(black) analysed, the frequency of RIP alleles and distribution of RIP mutations decreased in a 5′ to 3′gradient (arrows). The single copy region (149 bp) between NC3 and AvrLm6 was not analysed and so the gradient arrow is discontinuous. * denotes a repeat region (620 bp) directly upstream of NC3 that is highly RIP-affected.
Figure 2.
Quantitative reverse transcriptase (RT)-PCR analyses of AvrLm6, AvrLm1, LmCys1 and LmCys2.
At least 100 fold changes in expression were observed for each gene in planta compared to in vitro growth. RNA was prepared from seven day old cultures of isolates with the wild type alleles AvrLm6-0, AvrLm1-0, LmCys1-0 and LmCys2-0, which were growing in 10% V8 juice. Additionally RNA was prepared from cotyledons of B. napus cv. Beacon 7 dpi with the same isolates. Two isolates were used. Transcript levels of each gene were compared to those of L. maculans actin within each sample. Data points are the average expression of each gene relative to actin determined from the two isolates, and three technical replicates for each sample. Expression relative to actin is presented as a log scale (Y-axis). Standard errors are represented.
Table 4.
Nucleotide changes in non-coding, non-repetitive regions within the AvrLm1-LmCys2 genomic region in Australian isolates of Leptosphaeria maculans.
Figure 3.
Distribution of RIP mutations across the AvrLm1-LmCys2 genomic region in Leptosphaeria maculans.
(A) The ratio of mutated CpA or TpG sites within the seven AvrLm6 RIP alleles compared to the number of available CpA and TpG nucleotides present in the wild type AvrLm6-0 allele over a 100 bp window. Regions where greater than 50% of potential RIP sites are mutated are highlighted in red. There is a higher proportion of RIP mutations towards the 5′ end of the region (3′ end of the AvrLm6 gene). AvrLm6-0, -1, -2, -3 and -4 alleles do not display RIP mutations. (B) The average ratio of RIP mutations within the untranslated regions (UTRs), exons and introns for the seven RIP alleles of AvrLm6 relative to the number of potential RIP sites in the wild type sequence. The 3′ UTR and exons are undergoing the highest frequency of RIP. (C) The number of RIP alleles for each of the genes and non-coding, non-repetitive (NC) regions analysed across the AvrLm1-LmCys2 genomic region in 84 isolates (see also Figure S2). The number of RIP alleles is highest for NC3 and decreases in the genes and non coding regions downstream, consistent with RIP occurring in a directional manner.* represent RIP alleles of AvrLm6 that are not transcribed and that confer a virulence phenotype on an Rlm6-containing cultivar. The remaining RIP alleles of AvrLm6 were not tested for virulence towards Rlm6.
Table 5.
Expression analysis of AvrLm1, AvrLm6, LmCys1 and LmCys2 alleles in Leptosphaeria maculans isolates in planta.
Table 6.
Analysis for the presence of a molecular clock, and relative rates of sequence evolution for genes within the AvrLm1-LmCys2 genomic region of Leptosphaeria maculans.
Table 7.
Analysis of positive selection on amino acids of proteins encoded within the AvrLm1-LmCys2 genomic region of Leptosphaeria maculans.
Figure 4.
Genetic diversity and phylogenetic relationships between haplotypes of Leptosphaeria maculans.
Un-rooted strict consensus of 1000 ML trees (-ln L = 16381.75) constructed from the concatenated DNA sequences of the seven genes and four non-coding, non-repetitive regions of the AvrLm1-LmCys2 genomic region. All CpA to TpA and TpG to TpA nucleotide changes were removed from the data set prior to analysis. Haplotypes (see Table S6) associated with RIP alleles cluster in a distinct clade with high bootstrap support (99%), suggesting a single origin. In contrast, haplotypes related to deletion alleles (*) are associated with multiple clades suggesting multiple origins. Note that RIP associated alleles have much longer branches (i.e. larger genetic distance) due to an accelerated evolution compared to non-RIP alleles (see Table 6). Haplotype 42 (circled) has RIP alleles at five of the loci examined (NC3, AvrLm6, LmCys1, NC4 and LmTrans), resulting in an exceptionally long branch.
Table 8.
Hypothesis-testing of multiple (polyphyletic) vs. single (monophyletic) origin of RIP mutation-associated haplotypes of Leptosphaeria maculans.